Library of compounds labelled with radiosotope

ABSTRACT

Library of compounds or their pharmaceutically acceptable salts, each compound being associated with information on its chemical identity and structure, wherein at least two of the compounds is labelled with radioisotope characterised in that the radioisotope is an AMS active radioisotope; A solid support having a compound or its pharmaceutically acceptable salt as hereindefined bound thereto, the compound being associated with information on its chemical identity and structure and comprising a radioisotope, characterised in that the radioisotope is an AMS active radioisotope as hereinbefore defined; Process for the preparation of a library of compounds as claimed in any of claims  1  to  19  comprising radioisotope labelling a plurality of compounds, each compound being associated with information on its chemical identity and structure characterised in that labelling is with an AMS active radioisotope; A kit therefor; Method for selecting one or more candidate compounds comprising screening a library of the invention comprising AMS active radioisotope labelled compounds as hereinbefore defined and obtaining a sample from the screen or submitting a compound identified for metabolic studies and obtaining a sample therefrom, and performing AMS detection of the sample; and Use of the library, a solid support comprising radioisotope labelled compound or a method as hereinbefore defined in (bio)medical, agrochemical, environmental and like screening for further study by AMS detection.

The present invention relates to a library of compounds labelled withradioisotope for detection of individual compounds, a process for thepreparation thereof, a method for selecting from a library a candidatecompound displaying desired characteristics and detection thereof in asimultaneous or subsequent study, and the use thereof in compoundselection and detection; more particularly the invention relates to alibrary of compounds labelled with AMS (accelerator mass spectrometry)active radioisotope for detection of individual compounds, a process forthe preparation thereof, a method for selecting from a library acandidate compound displaying desired characteristics and detectionthereof by AMS; and the use thereof in compound selection, in particularin pharmaceutical drug screening, and AMS detection providing in vivometabolism characteristics thereof.

The process of drug discovery and development for the pharmaceutical andbiotechnology industries involves a host of different activitiesfollowing initial selection of a number of candidate drugs, Phase 1studies requiring scale up of drug production, preclinical toxicology,GMP manufacture, animal adsorption, diffusion, metabolism, excretion(ADME) studies etc. Before entering Phase 2 trials as many as one drugin three will have been dropped because of pharmacokinetic (PK),pharmacodynamic or toxicity issues. This process involves enormous costwhich is reflected in the high costs of pharmaceuticals brought tomarket. Moreover the high failure rate of drug candidates furtherincreases the cost of the successful candidates brought to market.

More recently new technologies have been adopted improving speed tomarket and improving initial drug candidate selection in the hope ofimproving the success rate during trials. For example selection of adrug may now be made from a larger number of candidates using highthroughput screening of candidates in trace amounts. Moreover thecandidate drugs screened may be taken from a chemical library comprisinghundreds or thousands of analogue chemicals obtained from acombinatorial chemistry approach. The combinatorial chemistry approachhas a further advantage in that a large number of compounds may bescreened, of which the structures need not be known, the libraryproviding structure information either in the form of a compound tag orcompound number. On selection of a number of candidates from the librarythey are then identified and forwarded for scale up of drug productionfor the next stage of trials.

In addition Accelerator Mass Spectrometry (AMS) is increasinglyreplacing the former in vitro techniques used to indicate in vivometabolism characteristics, giving massive improvements in accurateassessment of in vivo metabolism of compounds. This has led to thedevelopment of human microdosing (Human Phase 0) which is arevolutionary new concept which relies on the ultrasensitivity of theAMS technique.

In microdosing one or more drug candidates are taken into humans intrace doses in order to obtain early ADME and PK information. Thisinformation is then used as part of the process for selection ofsuitable drug candidates, to select which of the microdosed drugs hasthe appropriate PK parameters to take further. The low dose screeningADME studies ensure that drugs do not have to be dropped later down thedevelopment pathway because of inappropriate metabolism such as firstpass, too short a half life, poor bio-availability etc. Humanmicrodosing dramatically reduces attrition in drug candidate selectionat Phase 1 trials.

Using the AMS approach it is however necessary to provide a radiolabelled version of a candidate compound, after initial candidateselection by conventional means, and this requires a custom synthesis ofradioiosotope labelled candidates. Although microdosing means thatsynthesis need be only in microdose amounts, the need for customsynthesis and scale up nevertheless provides a bottleneck in theselection method and adds greatly to costs and delays. It is thereforedesirable to facilitate this stage to speed up the drug discoveryprocess and reduce costs.

We have now surprisingly found that it is possible to provide animproved libraries comprising compounds labelled with radioisotope foruse in candidate compound selection and subsequently determining thefate of the compound by detection thereof, for example detecting by itslocation on binding or detecting in vivo metabolism characteristicsassociated with individual library members. This eliminates the need forcustom radioisotope labelling of selected candidate drugs at a stage inthe selection procedure which effectively brings the entire selection toa halt pending the time consuming synthesis. Moreover the candidatecompounds lacking the necessary metabolism characteristics may beeliminated from the drug selection at a much earlier stage dramaticallyreducing attrition in the selection process.

In the broadest aspect of the invention there is therefore provided alibrary of compounds or their pharmaceutically acceptable salts, eachcompound being associated with information on its chemical identity. andstructure, wherein at least two of the compounds is labelled withradioisotope characterised in that the radioisotope is an AMS activeradioisotope.

AMS is used for the efficient detection of long-lived isotopes atpart-per-quadrillion sensitivities, and can analyse ¹⁴C at attomole tozeptomole levels (10⁻¹⁸-10⁻²¹ Moles). AMS performs liquid scintillationcounting of radioactive samples of body fluids obtained from a human whohas received radioactive doses. One of the most significant advantagesof AMS is that it can detect and quantify with relatively shortanalytical times, levels of radioactivity that are so low that the doseneeded to be administered to a human subject falls below the stipulatedlevels of radioactivity which require regulatory review.

AMS active radioisotopes include any isotopes which are susceptible toAMS analysis. AMS active radioisotopes preferably have low naturalbackgrounds, for example in the range from 1×10⁻⁵% or less for exampleto 1×10⁻¹⁵%. The sensitivity of AMS relies on the fact that AMS activeradioisotopes have a very low natural background such as 0.00000000001%for ¹⁴C. The background for ¹³C is 1.1%, which by comparison is huge.This means that the incorporation rate for ¹³C would have to be muchhigher than for ¹⁴C. AMS also generates and analyses negative ions,preferably therefore an AMS active radioisotope is able to form negativeions. Preferably AMS active radioisotopes have long half lives in excessof weeks up to 1,000's of years for ease of handling. Preferably AMSactive radioisotopes are non-toxic in AMS active; levels, whereby theyare suitable for human metabolism, and preferably are of biomedicalinterest.

The library of the invention may be envisaged for purpose of detectionin screening as hereinbefore defined, and comprise an AMS activeradioisotope directly incorporated in the compound as defined orassociated with the compound as a tag or the like. Preferably the AMSactive radioisotope is incorporated directly in library compounds and isnot detachable therefrom, except by degradation of the compound itself,for example by metabolic degradation. A radioisotope is thereforeassociated by means other than readily hydrolysed linkages or linkageswhich may be readily cleaved under acidic conditions or the like.

Preferably the radioisotope is covalently incorporated in the compound.More preferably the AMS active radioisotope is present as an atom makingup the chemical structure of a library compound, introduced orsubstituted as the desired radioactive isotope and is not incorporatedin a bead or tag associated with the compounds. Most preferably an AMSactive radioisotope is present as a cyclic ring atom or heteroatom,preferably an aromatic ring atom or heteroatom, or as an aliphaticcarbon chain atom or heteroatom such as a saturated or unsaturated atomor heteroatom, which may be single, double or triple bonded, or thelike.

The radioisotope thereby forms an integral part of the compound. Itshould be appreciate that this is distinct from a radioisotope labelapplied to a tag such as a support on which a compound is supported,such as a bead, or applied to an identifier tag to provide informationon the identity of, or a step in the synthesis of, a given compound.Radioisotope labelled compounds may nevertheless be envisaged for thelibrary of the invention,. in which the radioisotope is present on a tagor other component, which is resilient to metabolic cleavage, andthereby is AMS active.

Preferably the library comprises a plurality of compounds or theirpharmaceutically acceptable salts of formula I:

wherein each

is different and is a compound which comprises an AMS activeradioisotope *;

-   -   m is a value for the percent incorporation of radioisotope and        is fractional in the range from in excess of zero to 1%; and    -   t is a tag associated with information on the compounds chemical        identity and structure wherein n is 0, or a whole number        integer.

Preferably m is fractional and is in the range from in excess of zero to0.1% whereby each compound of formula I is lightly labelled, aproportion thereof having no radioisotope. Different compounds offormula I may comprise same or different radioisotope, and any onecompound of formula I may comprise same or different radioisotopepresent on the same or different molecules. A library is thereforesuitably present as a plurality of lightly labelled compounds; or may bepresent as a plurality of labelled compounds and a correspondingplurality of fully labelled compounds in lesser amount which may becombined to give a lightly labelled compound with appropriate %incorporation, as desired.

Preferably the proportion of radioisotope labelled compounds of any onecompound of formula I is such as to provide, in sampling that compound,a sample which is within the limits of AMS detection. Preferablytherefore the radioisotope is present in an amount which is within thelimits of AMS detection. In a particular advantage the library of theinvention relies on the ultrasensitivity of the AMS technology whereby alibrary of radioisotope labelled compounds may be provided for screeningand AMS detection in ultralow quantities or lightly labelled suitablefor analysing compounds from the library only in those reactions whichcan be analysed using the AMS method.

Reference herein to a compound being lightly labelled is to the compoundcomprising radioisotope present in AMS active amount, preferablycorresponding to a value for percent incorporation in a range ashereinbefore defined. Percent incorporation is a measure of maximumspecific activity, wherein 100% incorporation is defined as theincorporation of one radioisotope per molecule, taking a given amount ofsubstance, in which every molecule has one specified atom replaced withits radioactive equivalent.

Preferably percent incorporation is in the range 1×10⁻¹² to 0.1%, morepreferably 1×10⁻¹⁰ to 0.1%. The library of the invention therefore takesadvantage of the analytical power of AMS and the fact that AMS canuniquely be applied to trace radioisotope labelled libraries.

Percent incorporation cannot be derived directly but is calculated fromthe specific activity of the compound. The maximum specific activity isgiven in dpm/mmole and is the basic unit ofradioactivity—disintegrations per minute—being the number of nucleardisintegrations occurring, on average, every minute. However percentincorporation is a normalised function which can be compared for allradioisotopes, and is more instructive than the term of specificactivity which is dependent on radioisotope. For example ¹⁴C as an AMSactive radioisotope may be present in a compound of the library of theinvention in an amount giving dpm/mmol in the range 5 to 12, preferably7 to 10, for example in the range 7.3438 to 9.8562 (ANU sucrose). Thevalue for another isotope would be different.

Preferably therefore % incorporation for a given compound is determinedby equation Equ 1: $\begin{matrix}{{\%\quad{incorporation}\quad(m)} = \frac{100 \times {specific}\quad{activity}}{{maximum}\quad{specific}\quad{activity}}} & {{Equ}\quad 1}\end{matrix}$

For any radioisotope (based on one radioisotope per molecule,corresponding to 100% incorporation) the maximum specific activity isgiven by the equation Equ 2: $\begin{matrix}{\frac{\ln\quad 2}{t_{1/2}} \times N} & {{Equ}\quad 2}\end{matrix}$where ln2 is the natural log of 2(=0.6932)

-   t_(1/2) is the half-life of the radioisotope in minutes-   N is the number of atoms of the radioisotope in 1 mmole (moles    compound×6.0225×10²⁰)-   (Lappin, G and Garner, R C (2003) Ultra sensitive detection of    radiolabelled drugs and their metabolites using Accelerator Mass    Spectrometry. Chapter 11 in: Wilson, I D (ed) Bioanalytical    Separations Handbook of Separations Vol 4. Elsevier Science BV    Amsterdam)

Equation 2 takes no account of diminishing radioactivity (dpm value) dueto the half-life of the radioisotope over time (ie it calculates themaximum specific activity at time zero).

The following example calculates the maximum specific activity (based onone radioisotope per molecule) for ¹⁴C. The half-life of ¹⁴C is 5730years and so any diminishment of radioactivity over short periods oftime is negligible. In the following illustration units are as follows:

-   Bq=Becquerel=60 dpm-   kBq: 60×10³ dpm-   GBq: 60×10⁹ dpm-   kBq/mmole: maximum theoretical specific activity (based on one    radioisotope per molecule).-   mmole=10⁻³ mole-   amole=10⁻¹⁸ mole

From equation Equ 2: $\begin{matrix}{{\frac{0.6932}{\left( {5730 \times 365.3 \times 24 \times 60} \right)} \times 6.0225 \times 10^{\quad 20}} = {1.385 \times 10^{11}{dpm}}} \\{= {2.3083 \times 10^{6}{{kBq}/{mmole}}}}\end{matrix}$

Thus, the maximum theoretical specific activity for ¹⁴C is 2.3083GBq/mmole, (based on one radioisotope per molecule). This equals 100%incorporation.

Percentage incorporation for lightly labelled compounds according to thepresent invention is therefore suitably determined by the above equationEqu 1:

Taking a library compound (x22) having specific activity 81.6 dpm/mmolas example:

-   This is 0.00136 kBq/mmol

If 100% incorporation=2.3083×10⁶ kBq/mmol, then, using Equation 1 above,the % incorporation for compound x22 is 5.8×10⁻⁸%. This very low, levelof incorporation is well within the sensitivity of AMS. AMS measures anisotope ratio (¹²C:¹⁴C in the present example). In the case of theanalysis of the library compounds, the background level was 1.46 amoles¹⁴C/mg C. The isotope ratio for compound x22 was 766.49 amoles ¹⁴C/mg C(ie ca 524 times background). This level of sensitivity is more thanadequate for many applications such as ligand binding.

It is also feasible to administer library compounds to laboratoryanimals or humans. A typical human radioactive dose, if AMS is beingused as the detection method, is 7.4 kBq. As an example, if the dose ofthe library compound was 100 mg and the molecular weight was 350, thenthe specific activity would be 7.4 kBq/0.2857 mmole, or 25.9 kBq/mmole.If 100% incorporation=2.3083×10⁶ kBq/mmole, then 25.9 kBq/mmole=0.001%incorporation.

An AMS active radioisotope may be selected from any radioisotope whichis amenable to detection by AMS detection techniques. Radioisotopes varyin half life and thereby in radioactivity and enable detection insmaller or greater amounts whereby certain radioisotopes areparticularly suited for certain envisaged applications either by virtueof the chemical nature of the isotope or its radiation characteristics.Many atoms are capable of forming several different radioisotopes, ofwhich certain may be suited for some radiodetection techniques andcertain suited for other techniques. Preferably therefore the libraryspecifies the nature of the radioatom and the particular isotope(s)present to indicate suitability of libraries for an intended use. Forexample a library labelled with ¹²⁹I is useful for AMS detection whereasa library labelled with 131I although highly active is probably oflimited use. Similarly a library labelled with ¹⁴C is useful for AMSdetection whereas a library labelled with ¹³C is of widespread use inmany other radioactive techniques, such as NMR detection, for example asdisclosed in WO97/01098, but of no use in AMS analysis. Particularlyunsuitable radioisotopes fails to form negative ions, notablyradioisotopes of nitrogen.

Compounds in the library of the invention therefore suitably compriseAMS active radioisotopes selected from AMS active radioisotopes ofhydrogen, beryllium, carbon, aluminium, phosphorus, chlorine, calcium,manganese, iron, selenium, iodine, barium and lanthanides and actinidessuch as uranium or plutonium. Preferably a library of the inventioncomprises compounds radioisotope labelled with an isotope selected fromany isotopes that are amenable to AMS analysis, preferably selected fromany one or more of ²H, ³, the isotopes of Ba, ¹⁰Be, ¹⁴C, ¹⁷O, ¹⁸O, ²⁶Mg,²⁶Al, ³²Si, ³⁶Cl, ⁴¹Ca, ⁵⁵Fe, ⁵⁷Fe, ⁶⁰Fe, ⁵³Mn, ⁵⁵Mn, ⁷⁹Se and ¹²⁹I,²³⁶U, ²³⁹Pu most preferably selected from any one or more of ³H, ¹⁴C and³⁶Cl.

In a preferred embodiment the invention comprises a library of compoundsas hereinbefore defined characterised in that an AMS active radioisotopeis a ¹⁴C radioisotope; alternatively or additionally a ³⁶Cl or ³Hradioisotope.

A library of compounds according to the invention preferably comprises aplurality of compounds present in solid phase or liquid phase, typicallysolution phase, or mixtures thereof, each compound supported on a solidsupport or contained within a sealed vial or the like, as known in theart. Compound supports or carriers such as beads or the like to whichthe library of compounds and tags have been added facilitate screeningof the compounds bound to the bead. Alternatively a library may compriseunsupported compounds, removed from the bead and grouped singly or in aset of 10 to 100 to 1000 or more compounds for screening. A solid phaselibrary may comprise compounds supported on small definable solidsupports, commercially available as particles or beads, capillaries,hollow fibers, needles, solid fibers, etc. The solid supports may benon-porous or porous, deformable or hard, and have any convenientstructure and shape. In some instances, magnetic or fluorescent beadsmay be useful. The beads will generally be at least 10-2000 micron,usually at least 20-500 micron, more usually at least 50-250 micron indiameter.

Preferably solid supports which may be employed include cellulose beads,controlled-pore glass beads, silica gel, polystyrene beads, particularlypolystyrene beads cross-linked with divinylbenzene, grafted co-polymerbeads such as polyethyleneglycol/polystyrene, polyacrylamide beads,latex beads, dimethylacrylamide beads, particularly cross-linked withN,N′-bis-acryloyl ethylene diamine and comprisingN-t-butoxycarbonyl-.beta.-alanyl-N′-acryloyl hexamethylene diamine,composites, such as glass particles coated with a hydrophobic polymersuch as cross-linked polystyrene or a fluorinated ethylene polymer towhich is grafted linear polystyrene; and the like.

In the library of the invention each. compound is associated withinformation on its chemical identity and structure as hereinbeforedefined and as is commonly known in the art of libraries such ascombinatorial libraries. Compounds may also be associated withinformation on the nature of the radioisotope(s) present and the amount,for example the % incorporation or specific activity, thereof.Association may be by means of physical association by means for exampleof a tag comprising synthesis information or a synthetic memoryassociated with a bead or solid support on which a compound is(releasably) supported or with which a compound is associated, or may beby numbering or indexing the container or well for, or location of, eachcompound and providing reference information on the chemical identityand structure of each numbered or indexed compound. Using well platessimplifies mapping of compound identity.

It is known to provide information on chemical identity and structure ina number of ways, including tagging a support, such as a bead, withwhich a compound in a library is associated, with a radioisotope. Suchtags are however designed to be readily cleaved in order to performassays unsupported or to read the synthesis information. It should beappreciated that the present invention is distinct in that radioisotopeof the library of the invention is present as an integral part of eachlibrary compound and is not detachable therefrom, for example if not anintegral part of a library compound structure is associated with apharmaceutically compatible tag which is irreversibly associated withthe compound to the degree that dissociation takes place only ondegradation of the compound itself, for example by metabolicdegradation. A radioisotope is therefore associated by means other thanreadily hydrolysed linkages or linkages which may be readily cleavedunder acidic conditions or the like.

Preferably the library of compounds is provided as an array of compoundssuitable for use in high throughput screening and the like. An array maycomprise a plate such as a microtiter plate, cell array, vial or bottlearray, support matrix or plate, fibre optic array or the like as knownin the art of combinatorial chemistry or may comprise a plurality ofsupports or containers such as vials, bottles or the like whereinsupport or container or compound is labelled with an identifier or tagwhich identifies the compound as a component of a library of compoundsor which identifies the compound by chemical identity and structure. Alibrary comprising compounds present on solid support(s) such as beadsmay in some cases provide an advantage in terms of ease of preparationand accuracy of detection in that unincorporated radioisotope from thesynthesis remaining in the reaction mix may be simply washed awayavoiding any contamination in use of the library or exceeding %incorporation levels as hereinbefore defined.

The purpose of the compound tags is for decoding the reaction history ofthe compound. The product may then be produced in a large synthesis.Where the reaction history unequivocally defines the structure, the sameor analogous reaction series may be used to produce the product in alarge batch. Where the reaction history does not unambiguously definethe structure, one would repeat the reaction history in a large batchand use the resulting product for structural analysis. In some instancesit may be found that the reaction series of the combinatorial chemistrymay not be the preferred way to produce the product in large amounts.

In the library and method of the invention a tag may be retained with orseparated from the compound for AMS study; and may be decoded prior toAMS detection but is preferably not decoded until the AMS resultsindicate that it is selected as a candidate for further trials.

Compounds present in the library may be present in any desired amount.In a particular advantage the library of the invention comprisescompounds in small quantities of nanomoles or millimoles, typicallymilligrams, by virtue of the sensitivity of AMS detection techniques, upto moles or grams. Suitably compounds are present in same or differentamounts in the range of 0.1 microgram to 10 g, preferably 1 microgram to100mg, for example 10 microgram to 10 mg. This has a double costadvantage, since the conventional unlabelled library is typically notcheap, and isotopes are also not cheap, whereby radioisotope labellingof compounds in small amounts helps to limit the cost of the library. Inthe preferred embodiment of the invention as hereinbefore defined theAMS active library provides a means to reduce the quantities ofcompounds present in a compound library by the adoption of the AMSdetection technique, thereby reducing cost.

The library of the invention may comprise any desired number ofcompounds. Commonly libraries are provided wherein from 20 to millionsof compounds are present. Preferably the library of. the inventioncomprises from 5 to 5×10⁶ compounds, for example from 20 to 1,000,000for example greater than 25,000 or greater than 50,000 compounds, suchas greater than 200,000 compounds. Particular advantages are associatedwith larger libraries of in excess of 25,000 compounds, since thebenefits of directly subjecting selected compounds from the library toradiodetection techniques increase with the number of compounds to bedetected. On the other hand the cost of a library increases with thenumber of compounds in the library and therefore there are advantagesassociated with smaller libraries of from 20 to 25,000 compounds.

In one embodiment all or substantially all compounds in the librarycomprise an amount of radioisotope as hereinbefore defined. Compoundsmay comprise the same or different radioisotope. Preferably thereforethe library comprises compounds as hereinbefore defined associated withinformation on the radioisotope identity or identities of eachindividual library member, for example to enable correct AMS samplepreparation, in view of the different sample preparation techniquesrequired for each type of radioisotope in AMS.

In an alternative embodiment a proportion of the compounds in thelibrary comprise a radioisotope. This may be of advantage in the case ofa method for screening a library for a first selection of candidatecompounds providing a desired activity, reactivity, functionality or thelike, in which the method includes making a secondary selection fromthose compounds identifying as positive in terms of activity,reactivity, functionality or the like, and forwarding the secondaryselection for radiodetection in a microdosing technique as hereinbeforeand hereinbelow defined. In the alternative embodiment of the inventionthe secondary selection would simply be to select those compounds whichshow best activity and which comprise a radioisotope.

Suitably at least 40% of the compounds are lightly labelled, preferablyat least 50% of the compounds are lightly labelled, more preferably atleast 75% of the compounds are lightly labelled, more preferably atleast 90% of the compounds are lightly labelled, most preferably all orsubstantially all of the compounds are lightly labelled.

A compound of the library of the invention may comprise more than oneradioisotope which may be the same or different, and are preferablydifferent. In the method of microdosing and AMS detection ashereinbefore defined it is common for compounds to be metabolised invivo and indeed it is this metabolic data which is sought in the method.Preferably therefore the library of the invention comprises a pluralityof compounds having a plurality of radioisotopes introduced randomly orspecifically in different moieties of the compound, whereby themetabolic pathway for potentially active and potentially inert moietiesmay be monitored giving more comprehensive metabolic information on thetarget delivery sites of active and inert moieties.

The library may be structurally diverse or similar and is suitably achemical or a biochemical library. Preferably in one embodiment thelibrary comprises organic compounds which are not amenable tobiochemical synthesis, ie synthetically obtained non-biochemicalcompounds. In an alternative embodiment the library may comprisebiochemical compounds made up of individual units such as amino acids,peptides, nucleic acids, fatty acids, carbohydrates etc. Howeverparticular advantages are obtained with libraries other than peptidesand oligonucleotides.

The library may be a combinatorial library comprising compounds whichare analogues of a common structure obtained from a combinatorialsynthesis or biosynthesis; or dissimilar compounds having commonreactive functionality suitable for targetting a particular reaction ormechanism, or may be structurally diverse compounds providing multiplestructure types or reactive functionality suitable for targetting orprobing an unknown reaction or mechanism type.

Preferably the library of the invention comprises a plurality of smallmolecules, typically naturally occurring or synthetic chemical orbiochemical bioactive molecules and their analogues for example of up to1000 MW. Alternatively the library comprises a plurality of largermolecules, typically naturally occurring or synthetic biomolecules andtheir analogues, such as radioisotope labelled biopolymers includingradioisotope labelled recombinant proteins such as insulin analogues,growth hormone analogues, antibodies and the like, peptides, plant orgene therapy products.

In a further aspect of the invention there is provided a solid supporthaving a compound or its pharmaceutically acceptable salt bound thereto,the compound being associated with information on its chemical identityand structure and comprising a radioisotope, characterised in that theradioisotope is an AMS active radioisotope as hereinbefore defined.

A solid support is suitably any solid support as known in the art ofchemical libraries as hereinbefore defined, and is preferably associatedwith information on its chemical identity and structure as hereinbeforedefined. Preferably the solid support is characterised by furtherfeatures as hereinbefore and hereinbelow defined in respect of a libraryof the invention.

In a further aspect of the invention there is provided a process for thepreparation of a library of compounds as hereinbefore defined comprisingradioisotope labelling a plurality of compounds, each compound beingassociated with information on its chemical identity and structurecharacterised in that labelling is with an AMS active radioisotope.

The process may be a process for preparation of a solution phase orsolid phase library of compounds, unsupported or supported usingtechniques as known in the art. Preferably labelling is performed inmanner to provide further features of a library as hereinbefore defined.

Labelling may be conducted as part of any known single or multistepsynthetic route or biosynthesis, or may be conducted as a dedicatedchemical or biosynthetic labelling step on commercially available orpreviously synthesised compounds, or a mixture thereof. Radioisotopelabelling is suitably performed by techniques as known in the art forlabelling compounds. A biosynthesis is suitably performed by culturing amicrorganism which produces biochemical products in a radioisotopeenriched environment and harvesting labelled products. Preferably theenriched environment comprises an AMS active radioisotope ashereinbefore defined, preferably in AMS active amount as hereinbeforedefined. Biochemical components or metabolites or microorganisms becomelabelled as a result of growing the microorganism in the enrichedenvironment as known in the art.

Preferably the process comprises lightly labelling a synthetic precursoror intermediate or a biochemical culture substrate and reacting withother precursors or intermediates, or culturing a microrganism thereinwhereby the radioisotope is incorporated in the synthesis orbiosynthesis product. It is not always possible to control the percentincorporation of a radioisotope in a compound, or in this case in aprecursor, intermediate or culture substrate, whereby. Preferablytherefore the process comprises labelling a synthetic precursor orintermediate or a biochemical culture substrate, determining thespecific activity thereof, determining the desired specific activity togive a desired percent incorporation, and combining with a sufficientamount of corresponding unlabeled synthetic precursor or intermediate ora biochemical culture substrate and isolating as a homogeneous producthaving desired percent incorporation. Preferably isolating a homogeneousproduct is by recrystallisation of the combined labelled and unlabelledproduct.

Thereafter the synthesis or biosynthesis process using the lightlylabelled precursor or intermediate or biochemical culture substrateincorporates radioisotope in desired percent incorporation.

Processes and techniques are available for performing gram or milligramscale scale custom syntheses of radioisotope labelled compounds. It iswithin the expertise of the skilled person to combine such customsynthetic processes and techniques with combinatorial techniques toprovide combinatorial radioisotope syntheses.

Methods for preparing combinatorial libraries are known in the art andinclude the techniques of parallel or series synthesis, split pool orsplit and mix synthesis whereby intermediates are split for diversereactions and mixed for common reactions, and the like. Synthesis may becarried out in dedicated combinatorial reactors such as multi-reactorsynthesisers, or in conventional manner.

Radioisotope labelled combinatorial methods may therefore be envisagedin which a core molecule is radioisotope labelled, preferably lightlylabelled as hereinbefore defined, and split into a plurality of samples,each of which is then subject to combinatorial variation, by reactionwith a known or random, structured or diverse, collection ofderivatisation reagents in one or more stages to provide a library ofradioisotope labelled derivatives. Alternatively a core molecule may besplit into a plurality of samples, each of which is then subject tocombinatorial variation, by reaction with a known or random collectionof, preferably lightly, radioisotope labelled derivatisation reagents inone or more stages to provide a library of, preferably lightly,radioisotope labelled derivatives.

Compounds of the library of the invention may be obtained from reactionsinvolving modifications at a variety of random sites of a central coremolecular structure or modifications at a specific site, as known in theart. For example, one may brominate a polycyclic compound, wherebromination may occur at a plurality of sites or use a brominating agentwhich will be specific for a particular site, e.g., N-bromosuccinimide.For the most part, reactions will involve single sites or equivalentsites, for example, one of two hydroxyl groups of a glycol.

For the most part, compounds of the library of the invention may beobtained from a synthesis having at least two stages where other thanbifunctional compounds are attached using the same linkingfunctionality, e.g. amino acids and amide bonds, nucleotides andphosphate ester bonds, or mimetic compounds thereof, e.g.,aminoiso-cyanates and urea bonds. Preferably the process comprisesserial synthesis involving the addition or removal of chemical units,reactions involving the modification or introduction of one or morefunctionalities, ring openings, ring closings, etc. Chemical units cantake many forms, both naturally-occurring and synthetic, such asnucleophiles, electrophiles, dienes, alkylating or acylating agents,diamines, nucleotides, amino acids, sugars, lipids, or derivativesthereof, organic monomers, synthons, and combinations thereof.Alternatively, reactions may be involved which result in alkylation,acylation, nitration, halogenation, oxidation, reduction, hydrolysis,substitution, elimination, addition, and the like. Compounds may benon-oligomers, oligomers, or combinations thereof in extremely smallamounts, where the reaction history, and composition in appropriatecases, can be defined by the tags as known in the art. Non-oligomersinclude a wide variety of organic molecules, e.g. heterocyclics,aromatics, alicyclics, aliphatics and combinations thereof, comprisingsteroids; antibiotics, enzyme inhibitors, ligands, hormones, drugs,alkaloids, opioids, terpenes, porphyrins, toxins, catalysts, as well ascombinations thereof. Oligomers include oligopeptides, oligonucleotides,oligosaccharides, polylipids, polyesters, polyamides, polyurethanes,polyureas, polyethers, poly (phosphorus derivatives) e.g. phosphates,phosphonates, phosphoramides, phosphonamides, phosphites,phosphinamides, etc., poly (sulfur derivatives) e.g. sulfones,sulfonates, sulfites, sulfonamides, sulfenamides, etc., where for thephosphorous and sulfur derivatives the indicated heteroatom for the mostpart will be bonded to C, H, N, O or S. and combinations thereof.

Known combinatorial synthetic methods permit variation in reaction ateach stage, depending on the choice of agents and conditions involved.Thus, for amino acids, one may have up to 20 amino acids involved usingthe common naturally-encoded amino acids and a much wider choice, if onewishes to use other amino acids, such as D-amino acids, amino acidshaving the amino group at other than the alpha-position, amino acidshaving different substituents on the side chain or substituents on theamino group, and the like. For the different nucleic acids, there willusually be up to 4 natural nucleic acids used for either DNA or RNA anda much larger number is available if one does not choose to use thoseparticular nucleic acids. For the sugars and lipids, there are a verylarge number of different compounds, which compounds may be furtherincreased by various substitutions, where all of these compounds may beused in the synthesis. For individual organic compounds the choice maybe astronomically large. In addition, one may have mimetic analogues,where ureas, urethanes, carbonylmethylene groups, and the like maysubstitute for the peptide linkage; various organic and inorganic groupsmay substitute for the phosphate linkage; and nitrogen or sulfur maysubstitute for oxygen in an ether linkage or vice versa.

The library of the invention may be obtained by a synthetic strategywhich varies with the nature of the group of products one wishes toproduce. Thus, the strategy must take into consideration the ability tostage-wise change the nature of the product, while allowing forretention of the results of the previous stages and anticipating needsfor the future stages. Where the various units are of the same family,such as nucleotides, amino acids and sugars, the synthetic strategiesare relatively well-established and frequently conventional chemistrywill be available. Thus, for nucleotides, phosphoramidite or phosphitechemistries may be employed; for oligopeptides, Fmoc or Boc chemistriesmay be employed where conventional protective groups are used; forsugars, the strategies may be less conventional, but a large number ofprotective groups, reactive functionalities, and conditions have beenestablished for the synthesis of polysaccharides. For other types ofchemistries, one will look to the nature of the individual unit andeither synthetic opportunities will be known or will be devised, asappropriate.

In some instances, a library of the invention may comprise compoundshaving the same or different blocks introduced at the same or differentstages in the synthesis. For example, one may wish to have a commonpeptide functional unit, e.g. the fibronectin binding unit (RGDS), apolysaccharide, e.g. Lex, an organic group, e.g. a lactam, lactone,benzene ring, olefin, glycol, thioether, etc. introduced during thesynthesis. In this manner one may achieve a molecular context into whichthe variation is introduced. These situations may involve only a fewstages having the plurality of choices, where a large number of productsare produced in relation to a particular functional entity. This couldhave particular application where one is interested in a large number ofderivatives related to a core molecule or unit known to have acharacteristic of interest.

In one embodiment the library of the invention is preferably obtained bybatch synthesis of a few compounds which would be prepared during thecourse of the combinatorial synthesis. By taking extreme examples, forexample, syntheses which might involve steric hindrance, charge and/ordipole interactions, alternative reaction pathways, or the like, one canoptimise conditions to provide for enhanced yields of compounds whichmight not otherwise be formed or be formed only in low yield. In thismanner, one may allow for a variety of reaction conditions during thecombinatorial synthesis, involving differences in solvent, temperatures,times, concentrations, and the like. Furthermore, one may use the batchsyntheses, which will provide much higher concentrations of particularproducts than the combinatorial synthesis, to develop assays tocharacterise the activity of the compounds.

Preferably the method comprises the synthesis of a single or mixedsolution-phase/solid-phase lightly labelled library. incorporating tracelevels of ¹⁴C lightly radioisotope labelled precursor. Preferablyprecursors are core labelled not substituent labelled, for examplelightly ring labelled benzoic acid.

Preferably the method comprises a 2 to 6 component condensation,substitution or the like reaction as hereinbefore defined, for example afour-component condensation such as an Ugi reaction ((a) Cao, X; Moran,E. J.; Siev, D.; Lio, A.; Ohashi, C.; Mjalli, A. M. M. Bioorg. & Med.Chem. Lett., 1995, 5, 2953-2958 and (b) Nakamura, M.; Inoue, J.; Yamada,T. Bioorg. & Med. Chem. Lett., 2000, 10, 2807-2810). This condenses acarboxylic acid, amine, aldehyde and isocyanide to form a substitutedalpha (acylamino) amide. In Scheme 1 and 2, any one or more of the fourcomponents may be lightly labelled, with the same or different AMSactive radioisotope.

For a solid-phase library preferably a solid-supported precursor is usedeg an amine (scheme 2).

Test runs may be undertaken on representative library members usingunlabelled (‘cold’) precursor, eg benzoic acid. The results indicatelibrary members containing benzaldehyde as a building block that cannotbe synthesised and as such these may be removed from the library.

A biochemical library is conveniently prepared by growing microorganismsin an AMS active radioisotope enriched environment as hereinbeforedefined. Known techniques include growth of bacteria or yeast in thepresence of labelled carbohydrate and salts, or in labelled methanol, orin labelled algal lysates, phototrophic culture of algae in labelledCO₂, growth of mammalian or insect cells in labelled media, and thelike. Any components of the microorganism can be harvested as lightlylabelled precursor or library compound, for example amino acids, fattyacids, carbohydrates, nucleic acids etc.

Preferably microorganisms are grown in lightly radioisotope labelledculture such as ¹⁴C glucose, and encouraged to mutagenise formingmutated bacteria, plated and cultured to form colonies generatingsecondary metabolites which are radioisotope labelled, and metabolitesare harvested providing a library of the invention. Harvesting may be bydisrupting the culture and lysing the bacteria, or by lifting offexcreted metabolites.

The library of the invention may be provided on any known supporttypical of libraries as known in the art as hereinbefore defined whichcan be readily mixed, separated, and serve as a solid substrate for thesequential synthesis. Depending upon the nature of the synthesis, thebeads may be functionalised in a variety of ways to allow for attachmentof the initial reactant. These may be linked through a non-labilelinkage such as an ester bond, amide bond, amine bond, ether bond, orthrough a sulfur, silicon, or carbon atom, depending upon whether onewishes to be able to remove the product from the bead. Conveniently, thebond to the bead may be permanent, but a linker between the bead and theproduct may be provided which is cleavable such as exemplified inTable 1. Two or more different linkages may be employed to allow fordifferential release of tags and/or products.

Depending upon the nature of the linking group bound to the particle,reactive functionalities on the bead may not be necessary where themanner of linking allows for insertion into single or double bonds, suchas is available with carbenes and nitrenes or other highly-reactivespecies. In this case, the cleavable linkage will be provided in thelinking group which joins the product or the tag to the bead.

Desirably, when the product is permanently attached, the link to thebead will be extended, so that the bead will not sterically interferewith the binding of the product during screening. Various links may beemployed, particular hydrophilic links, such as polyethyleneoxy,saccharide, polyol, esters, amides, combinations thereof, and the like.

Functionalities present on the bead may include hydroxy, carboxy,iminohalide, amino, thio, active halogen (Cl or Br) or pseudohalogen(e.g., —CF₃, —CN, etc.), carbonyl, silyl, tosyl, mesylates, brosylates,triflates or the like. In selecting the functionality, someconsideration should be given to the fact that the identifiers willusually also become bound to the bead. Consideration will includewhether the same or a different functionality should be associated withthe product and the identifier, as well as whether the twofunctionalities will be compatible with the product or identifierattachment and tag detachment stages, as appropriate. Different linkinggroups may be employed for the product, so that a specific quantity ofthe product may be selectively released. In some instances the particlemay have protected functionalities which may be partially or whollydeprotected prior to each stage, and in the latter case, reprotected.For example, amino may be protected with a carbobenzoxy group as inpolypeptide synthesis, hydroxy with a benzyl ether, etc.

Tags may be released from the library compound, and then subjected to adetecting means for example reacting with a molecule which allows fordetection. Such tags may be quite simple, having the same functionalityfor linking to the library compound as to the detecting means. Forexample, by being linked to a hydroxycarboxyl group, a hydroxyl groupwould be released, which could then be esterified or etherified with themolecule which allows for detection. For example, by using combinationsof fluoro- and chloroalkyl groups, in the binary mode, the number offluoro and/or chloro groups could determine choice, while the number ofcarbon atoms would indicate stage. Preferably the library of theinvention comprises compounds having detachable tags, for which thereare numerous functionalities and reactants known in the art.Conveniently, ethers may be used, where substituted benzyl ether orderivatives thereof, e.g. benzhydryl ether, indanyl ether, etc. may becleaved by acidic or mild reductive conditions. Alternatively, one mayemploy beta-elimination, where a mild base may serve to release theproduct. Acetals, including the thio analogues thereof, may be employed,where mild acid, particularly in the presence of a capturing carbonylcompound, may serve. By combining formaldehyde, HCl and an alcoholmoiety, an .alpha.-chloroether is formed. This may then be coupled withan hydroxy functionality on the bead to form the acetal. Variousphotolabile linkages may be employed, such as o-nitrobenzyl,7-nitroindanyl, 2-nitrobenzhydryl ethers or esters, etc. Esters andamides may serve as linkers, where half-acid esters or amides areformed, particularly with cyclic anhydrides, followed by reaction withhydroxyl or amino functionalities on the bead, using a coupling agentsuch as a carbodiimide. Peptides may be used as linkers, where thesequence is subject to enzymatic hydrolysis, particularly where theenzyme recognises a specific sequence. Carbonates and carbamates may beprepared using carbonic acid derivatives, e.g. phosgene, carbonyldiimidazole, etc. and a mild base. The link may be cleaved using acid,base or a strong reductant, e.g., LiAlH₄, particularly for the carbonateesters.

In a further aspect of the invention there is provided a kit forpreparing a library of the invention as hereinbefore defined, with themethod of the invention as hereinbefore defined, comprising one or moresets of a plurality of separated reactants, and optionally an amount ofone or more common reactants to be reacted with each set, each of thereactants characterised by having a distinguishable composition, beingassociated with information on structure or identity, and sharing atleast one common functionality, at least one set or one common reactantbeing labelled with an AMS active radioisotope as hereinbefore defined.

A kit may provide various reagents for use as tags in carrying out thelibrary syntheses. Reagents for use as tags may comprise at least 4,usually 5, different compounds in separate containers, more usually atleast 10, and not more than about 100, more usually not more than about36 different separated organic compounds. For binary determinations, themode of detection will usually be common to the compounds associatedwith the analysis, so that there may be a common chromophore, a commonatom for detection, etc. Where each of the identifiers is pre-prepared,each will be characterised by having a distinguishable compositionencoding choice and stage which can be determined by a physicalmeasurement and including groups or all of the compounds sharing atleast one common functionality.

Alternatively, the kit may provide reactants which can be combined toprovide the various identifiers or tags. Reactants may comprise aplurality of separated first functional, frequently bifunctional,organic compounds, usually four or more, generally one for each stage ofthe synthesis, where the functional organic compounds share the samefunctionality and are distinguishable as to at least one determinablecharacteristic. In addition, the kit may comprise at least one, usuallyat least two, second organic compounds capable of reacting with afunctionality of the functional organic compounds and capable of formingmixtures which are distinguishable as to the amount of each of thesecond organic compounds. For example, reagents may comprise a glycol,amino acid, or a glycolic acid, where the various bifunctional compoundsare distinguished by the number of fluorine or chlorine atoms present,to define stage, and have an iodomethane, where one iodomethane has noradioisotope, another has ¹⁴C and another has one or more ³H. By usingtwo or more of the iodomethanes, one could provide a variety of mixtureswhich could be determined by their radioemissions. Alternatively, onecould have a plurality of second organic compounds, which could be usedin a binary code.

In a further aspect of the invention there is provided a method forselecting one or more candidate compounds for medical applications,comprising screening a library of the invention comprising AMS activeradioisotope labelled compounds as hereinbefore and obtaining a samplefrom the screen or submitting a compound identified for metabolicstudies and obtaining a sample therefrom, and performing AMS detectionof the sample. Screening may be for a desired activity, reactivity,inhibition, functionality or the like, as known in the art, identifyingone or more candidate radioisotope labelled compounds from the library.AMS detection is suitably conducted on a screening sample or by dosing,for example microdosing, the candidate radioisotope labelled compoundsin human, animal or plant subjects and performing AMS detection ofmetabolic samples taken from the subjects. A sample is preferablyprepared for AMS from any sample which is derived from a screen, such asa cell or cell membrane sample, or from human, animal or plant deriveddosing samples, such as tissues or cells, bodily fluids such as blood orurine, faeces, plant tissues, soil or soil organisms such as worms andthe like.

The method of the invention is therefor useful both in providing for invitro activity, reactivity, inhibition or functionality screening andselection of compounds and in providing binding or in vivo metabolicdata for the selected candidate compounds, in particular for providingADME and PK data.

Screening is performed in known manner by taking a sample of eachcompound present in the library and subjecting to a desired assay.

Screening may be with any known or novel medical, biological,environmental or like screen and is typically a human or animalbiomedical assay or the like, for example a protein binding assay, suchas a receptor binding assay.

A wide variety of assays and techniques are commercially available todetermine a characteristic of interest of a screened compound.

Screening may be conducted on compounds associated directly with theiridentifiers, such as beads as hereinbefore defined, and may be conductedon single beads or groups of compounds to determine whether the compoundor groups show activity. Groups may involve 10, 100, 1000 or morecompounds. In this way, large groups of compounds may be rapidlyscreened and segregated into smaller groups of compounds.

A common screen is to detect binding to a particular biomolecule such asa receptor. The receptor may be a single molecule, a molecule associatedwith a microsome or cell, or the like. Where agonist activity is ofinterest, one may wish to use an intact organism or cell, where theresponse to the binding of the subject product may be measured. In someinstances, it may be desirable to detach the compound from the bead,particularly where physiological activity by transduction of a signal isof interest. Where binding is of interest, one may use a labeledreceptor where the label is a fluorescer, enzyme, radioisotope, or thelike, where one can detect the binding of the receptor to the compoundon the bead. Alternatively, one may provide for an antibody to thereceptor, where the antibody is labeled, which may allow foramplification of the signal and avoid changing the receptor of interest,which might affect its binding to the product of interest. Binding mayalso be determined by displacement of a ligand bound to the receptor,where the ligand is labeled with a detectable label.

A screen may comprise a two-stage screen, comprising binding as aninitial screen, followed by biological activity with a viable cell in asecond screen. Using recombinant techniques to prepare libraries allowsgreat variation in the genetic capability of cells. One can then produceexogenous genes or exogenous transcriptional regulatory sequences, sothat binding of gene or sequence to a surface membrane protein willresult in an observable signal, e.g. an intracellular signal. Forexample, a second screen may comprise introducing a leuco dye into thecell, where an enzyme which transforms the leuco dye to a coloredproduct, particularly a fluorescent product, becomes expressed uponappropriate binding to a surface membrane, e.g. beta-galactosidase anddigalactosidylfluorescein. In this manner, by associating a particularcell or cells with a particular candidate compound, the fluorescentnature of the cell may be determined using a FACS, so that activecandidate compounds may be identified. Various techniques may beemployed to ensure that the candidate compound remains bound to thecell, even where the product is released from the candidate compound.For example, the compound may comprise antibodies to a surface membraneprotein, eg one may link avidin to the surface of the cell and havebiotin linked to the candidate compound directly or via its carrier orbead, etc.

Assays may be performed stagewise using individual compounds or groupsof compounds or combinations thereof. For example, after carrying outthe combinatorial syntheses, groups of about 50 to 10,000 compounds maybe segregated in separate vessels. In each vessel a portion of the eachcompound is released, if bound to a carrier. The fractional release maybe as a result of differential linking of the product to the particle orusing a limited amount of a reagent, condition or the like, so that theaverage number of compound molecules released per carrier is less thanthe total number of compound molecules per carrier. The screen mediathen comprises a mixture of compounds in a small volume. The mixturecould then be used in an assay for binding, where the binding eventcould be inhibition of a known binding ligand binding to a receptor,activation or inhibition of a metabolic process of a cell, or the like.Various assay conditions may be used for the detection of bindingactivity as known in the art. Once a group is shown to be active, theindividual compounds may then be screened, by the same or a differentassay, giving a three- or four-stage procedure in total, where largegroups are divided up into smaller groups, etc. and finally singlecompounds are screened. In each case, portions of the compounds oncarriers would be released and the resulting mixture used in anappropriate assay. Assays may be the same or different, the moresophisticated and time consuming assays being used in the later or laststage.

Screening may alternatively be performed on spatial arrays, wherebycompounds may be distributed over a honeycomb plate, with each well inthe honeycomb having 0 or 1 compound.

Screening may be used to identify compounds with catalytic properties,such as hydrolytic activity, e.g. esterase activity. In a catalyticscreen compounds may be embedded in a semisolid matrix surrounded bydiffusible test substrates. If the catalytic activity can be detectedlocally by processes that do not disturb the matrix, for example, bychanges in the absorption of light or by detection of fluorescence dueto a cleaved substrate, compounds in the zone of catalytic activity canbe isolated and their identifier tags decoded.

Screening may be used to identify compounds with inhibitory oractivating activity. Compounds may be sought that inhibit or activate anenzyme or block a binding reaction. To detect compounds that inhibit anenzyme compounds are suitably released from carriers enabling them todiffuse into a semisolid matrix or onto a filter where this inhibition,activation or blocking can be observed. Compounds that form a visualisedor otherwise detectable zone of inhibition, activation or blocking canthen be picked and the tags decoded.

Tagging in this case is preferably by attached to the compounds bycleavable linkages, preferably a photolabile linkage, while a portion ofthe tags remain attached to the bead, releasable after picking by adifferent means than before.

A dialysis membrane may be employed where a layer of supported compoundsis separated from a layer of radioisotope labeled ligand/receptor pair.The compound layer may be irradiated with ultraviolet light releasingthe compound which would diffuse to the pair layer, where theradioisotope labelled ligand would be released in proportion to theaffinity of the compound for the receptor. The radioisotope labelledligand would diffuse back to the layer of compounds. Since theradioisotope would be proximal to the compound, compounds associatedwith radioemission would be analysed.

A screen may be used to identify compounds having biological activity.In some applications it is desirable to find a compound that has aneffect on living cells, such as inhibition of microbial growth,inhibition of viral growth, inhibition of gene expression or activationof gene expression. Screening of supported compounds may be achieved,for example, by embedding the supports in a semisolid medium and thelibrary of compounds released from the embedded supports enabling thecompounds to diffuse into the surrounding medium. The effects, such asplaques within a bacterial lawn, can be observed. Zones of growthinhibition or growth activation or effects on gene expression can thenbe visualised and compounds at the centre of the zone picked andanalysed.

A screen may include gels where the molecule or system, e.g. cell, to beacted upon may be embedded substantially homogeneously in the gel.Various gelling agents may be used such as polyacrylamide, agarose,gelatin, etc. Compounds may then be spread over the gel so as to havesufficient separation between the compounds to allow for individualdetection. If the desired compound is to have hydrolytic activity, asubstrate may be present in the gel which would provide a fluorescentproduct, enabling screening the gel for fluorescence and mechanicallyselecting compounds associated with the fluorescent signal.

Cells may be embedded in the gel, in effect creating a cellular lawn.Compounds may be spread out as described above. Techniques are known inthe art for placing a grid over a gel defining areas of one or nocompound. Cytotoxicity may be detected by releasing a library compound,incubating for a sufficient time, followed by spreading a vital dye overthe gel. Those cells which absorbed the dye or did not absorb the dyecould then be distinguished.

As known in the art cells can be genetically engineered so as toindicate when a signal has been transduced. There are many receptors forwhich the genes are known whose expression is activated. By inserting anexogenous gene into a site where the gene is under the transcriptionalcontrol of the promoter responsive to such receptor, an enzyme can beproduced which provides a detectable signal, e.g. a fluorescent signal.A library compound associated with the fluorescent cell(s) may then beanalysed for its reaction history.

The method of the invention includes selecting one or more compounds,for example 5 to 100 compounds in a successful screen, providing aradioisotope labelled sample of the selected compounds from the libraryof the invention and forwarding for radiodetection in a subsequentstudy, for example for AMS detection in a metabolic, pharmacokinetic orlike study.

AMS microdosing is suitably by administering an amount of candidatecompound alone or with a suitable carrier to a human or animal subject.Administration is typically by oral, dermal, buccal, vaginal, anal,subcutaneous, nasal route or by inhalation. A microdose suitablycomprises sufficient compound to give a low dose of the order ofnanocuries of radioactive label, for example is of the order of ng ormg. Preferably a microdose comprises 1-5 nanoCuries, more preferably isless than 1 microSievert, thereby being exempt from regulatory approval.A microdose may therefore comprise from 1 microgram to 1 milligram,preferably 1 microgram to 500 micrograms of radioisotope labelledcompound of the library of the invention.

After a period of days, weeks or months, samples are taken of tissue orcells, blood samples, urine or faeces. Samples are suitably taken atintervals in order to detect compound metabolism rate and indicate rapidand slowly metabolised compounds. The method is described in WO01/59476, the contents of which are incorporated herein by reference.

Analysis of AMS results indicates number of isotope counts, eg of ¹⁴C,ratio of modern (ie naturally occurring) isotopes and percent modernisotope as a combination of the number of counts and the ratio of modernisotope. pMC (percent modern carbon) is an AMS term of radioactivity andprovides a measure of the carbon content of a sample. pMC=Timesmodern×100. One times modern=¹⁴C/¹²C ratio in the atmosphere in 1952.The ratio ¹²C/¹³C remains relatively constant.

In the method of the invention a sample is prepared for AMS analysis ina range of micrograms or less of tissues or cells to a few microlitresof blood or urine. Samples may also comprise plant tissues, soil or soilorganisms such as worms, as known in the art.

The sample is prepared in a form that can yield negative ions within theinstruments ion source, as known in the art. Sample preparation may beby traditional methods which prepare thermally and electricallyconductive solids, are non fractionating, efficient and protected fromcontamination by isobars or unexpected concentrations of the rareisotope in or on laboratory equipment. Uniformity and comparabilitybetween samples and standards are ensured by reducing all samples to ahomogeneous state from which the final target material is prepared.Reduced sample is then compressed into tablet form in a cylindricalaluminium cathode before elemental isotope ratio analysis in the AMS.

For example samples obtained from microdosing isotopic carbon labelledlibrary compounds may be converted to graphite, samples obtained frommicrodosing isotopic halide labelled library compounds may be convertedto silver halide salts, samples obtained from microdosing isotopicaluminium labelled library compounds may be converted to aluminium oxideand samples obtained from microdosing isotopic calcium labelled librarycompounds may be converted to a calcium dihalide or dianhydride.Conversion is for example performed for carbon samples (containing ¹⁴C)by oxidising to CO₂ before reducing to graphite, commonly by thereduction of the CO₂ by hydrogen or zinc over an iron or cobalt catalystor binder (Vogel J S (1992) Rapid production of graphite withoutcontamination for biomedical AMS, Radiocarbon, 34, 344-350). Oxidationis in a sealed tube which is heated in a furnace at temperatures of upto 900 C. with an oxidant such as copper oxide for approx 8 hours. Theresulting CO₂ is reduced to graphite in a second step after cryogenictransfer using a reducing agent such as zinc and titanium hydride andcobalt as a catalyst at temperatures up to about 500 C. for approx 18hours with cooling. Cobalt/graphite is then compressed into tablet formin a cylindrical aluminium cathode before elemental isotope ratioanalysis in the AMS.

Alternatively sample preparation may be for example by the improvedtechnique of WO 01/59476, the contents of which are incorporated hereinby reference. Preferably according to the method of WO 01/59476 sampleis homogeneously mixed with a binder which is preferably electricallyconductive and may be any substance which allows the mixture of sampleand binder to be compressed into tablet form. More preferably the binderis one or a mixture of any of graphite, cobalt or aluminium powder, forexample where the isotope to be detected is ¹⁴C, or is one or a mixtureof any or aluminium oxide and iron or iron oxide, for example where theisotope to be detected is plutonium.

Preferably the method of the invention comprises in a further stageanalysing the results of AMS detection and identifying one or morecandidate compounds characterised by a desired metabolic profile in adesired subject and forwarding the identified candidate compound(s) forfurther studies on medical acceptability or efficacy.

In a further aspect of the invention there is provided a method for AMSdetection of isotope obtained from one or more radioisotope labelledcompounds present in samples of fluids taken from one or more human,animal or plant subjects dosed with one or more candidate compoundsidentified from a library as hereinbefore defined.

In a further aspect of the invention there is provided the use of alibrary, a solid support comprising radioisotope labelled compound or amethod as hereinbefore defined in (bio)medical, agrochemical,environmental and like screening for further study by AMS detection.

Preferably wherein (bio)medical screening is for compound activity,reactivity such as binding, inhibitory effect or other functionality, toassess for metabolism characteristics; agrochemical screening is forcompound activity, reactivity such as binding, inhibitory effect orother functionality, and assessing for plant, insect or like metabolism;environmental screening is for compound activity, reactivity such asbinding, inhibitory effect or other functionality, and assessing forsoil, aqueous or sediment absorption or adsorption, diffusion, leaching,metabolism, degradation, dissipation or photolysis study.

The library of the invention is useful in any applications in whichcompound libraries are currently used, wherein the analysis ofradioisotopes facilitates detecting the presence of a compound in asample, location of a compound for example by origin of sample, or theamount of a compound in any location or sample, using AMS radiodetectiontechniques. This may be of use during the initial screening of a libraryof compounds for example indicating successful binding to a desiredsubstrate. Alternatively or additionally the library of the inventionmay be of use after screening and selection of compounds having adesired activity, for example having a desired binding characteristic,in providing radioisotope labelled samples of selected librarycompounds, shown in an initial screen of the library to be activecompounds, for directly performing further studies requiring thepresence of radioisotopes, such as radiodetection of metabolic samples.

Preferably the library of the invention is for use in a method ofscreening for selecting candidate compounds and providing radioisotopelabelled samples of those compounds for determining binding to receptorsin cells, animal studies, investigating mechanism of action ofmetabolites, metabolic studies and the like, in known manner. Forexample receptor binding may be screened for a number of radioisotopicmetabolites and receptor-ligand complexes formed may be harvested andsubject to AMS to determine whether radiosotope is present indicatingreceptor binding by the library metabolite in question; or a screen maybe conducted for selecting candidate compounds for medical applicationsand dosing, preferably by microdosing, the candidate compounds in humanor animal subjects followed by AMS detection of samples of fluids takenfrom the subjects to determine metabolism characteristics. The libraryof the invention is therefore useful in providing in vivo metabolic datarelating to metabolism characteristics for the candidate compounds in amodular approach, without the need for intermediate determination ofcandidate and its synthesis, and synthesis of a radioisotope labelledanalogue.

The invention is now illustrated in non limiting manner with referenceto the following examples and Figures wherein:

FIGS. 1 to 5 show reaction schemes and structures of library compounds.

EXAMPLE 1 Synthesis of Solution Phase/Solid Phase Library IncorporatingTrace Levels of ¹⁴C Ring Labelled Benzoic Acid

A classical example of the production of a chemical library isillustrated by the Ugi reaction (Cao, X., Moran, E. J., Siev, D., Lio,A., Ohashi, C. and Mjalli, A M M (1995). Bioorg and Med Chem Lett 52953-2958 and Nakamura, M., Inoue, J. and Yamada, T. (2000) Bioorg andMed Chem Lett 10 2807-2810). The reaction, which can be conducted inliquid or on immobilised resin, consists of the condensation of fourreactants, in the current case a carboxylic acid, an amine, an aldehydeand an isocyanide. The end products (ie library compounds) are varied byselection of different reactants.

Following some exploratory experiments, the reactant mixtures were usedas described in Table 1. Reactions x1-x19 were conducted in solution.Reactions x21-x29 were conducted using TentaGel S-RAM resin. This solidsupport donates amine groups into the reaction. TABLE 1 Components usedfor the Ugi reaction. End pro- ¹⁴C duct benzoic code* Amines Aldehydesacid Isocyanides Solution based reaction x1 Benzylamine Isobutyraldehyde✓ Cycohexyl isocyanide x2 Benzylamine Valeraldehyde ✓ Cycohexylisocyanide x3 Benzylamine Cyclopentane ✓ Cycohexyl isocyanidecarboxaldehyde x4 Benzylamine Cyclohexane ✓ Cycohexyl isocyanidecarboxaldehyde x6 Benzylamine Isobutyraldehyde ✓ 2-morpholinoethylisocyanide x7 Benzylamine Valeraldehyde ✓ 2-morpholinoethyl isocyanidex8 Benzylamine Cyclopentane ✓ 2-morpholinoethyl carboxaldehydeisocyanide x9 Benzylamine Cyclohexane ✓ 2-morpholinoethyl carboxaldehydeisocyanide x11 Butylamine Isobutyraldehyde ✓ Cycohexyl isocyanide x12Butylamine Valeraldehyde ✓ Cycohexyl isocyanide x13 ButylamineCyclopentane ✓ Cycohexyl isocyanide carboxaldehyde x14 ButylamineCyclohexane ✓ Cycohexyl isocyanide carboxaldehyde x16 ButylamineIsobutyraldehyde ✓ 2-morpholinoethyl isocyanide x17 ButylamineValeraldehyde ✓ 2-morpholinoethyl isocyanide x18 Butylamine Cyclopentane✓ 2-morpholinoethyl carboxaldehyde isocyanide x19 Butylamine Cyclohexane✓ 2-morpholinoethyl carboxaldehyde isocyanide Immobilised based reactionx21 On resin Isobutyraldehyde ✓ Cycohexyl isocyanide x22 On resinValeraldehyde ✓ Cycohexyl isocyanide x23 On resin Cyclopentane ✓Cycohexyl isocyanide carboxaldehyde x24 On resin Cyclohexane ✓ Cycohexylisocyanide carboxaldehyde x26 On resin Isobutyraldehyde ✓2-morpholinoethyl isocyanide x27 On resin Valeraldehyde ✓ x28 On resinCyclopentane ✓ 2-morpholinoethyl carboxaldehyde isocyanide x29 On resinCyclohexane ✓ 2-morpholinoethyl carboxaldehyde isocyanide*code numbers are not necessarily in numerical order. The end productsshown were chosen following preliminary trial experiments and not allreaction mixtures formed the desired product. This is not uncommon withUgi reactions.

The reactions and end products are shown in FIGS. 1 to 5.

1A—Preparation of Radiolabelled Precursor

The carboxylic acid, which was constant for all reactions, was labelledwith ¹⁴C. Non-radiolabelled benzoic acid (ca 1.5 g) was dissolved in hotwater along with ca 4,200 dpm of ¹⁴C-ring labelled benzoic acid (Sigma)followed by recrystallisation by cooling (yield ca 97% lightly labelled¹⁴C ring labelled benzoic acid).

1B—General Procedure for Solution Phase Synthesis

To anhydrous methanol (0.5 ml) was added amine (0.4 mmol) and aldehyde(0.4 mmol). The resulting solution was agitated for 10 min at 28° C. Tothis solution was added ¹⁴C lightly ring labelled benzoic acid (from 1A)(0.4 mmol) in anhydrous methanol (1 ml) followed by isocyanide (0.4mmol). The reaction was agitated for 60 h at 28° C. For all reactionsexcept x4 the mixture was evaporated in vacuo and the residueredissolved in ethyl acetate (10 ml) and saturated NaCl (10 mnl), driedover MgSO₄ and filtered. The organic layer was evaporated in vacuo toyield the crude condensation product.

Following completion of the reaction, the products were characterisedwith mass-spectroscopy (the results are shown in FIGS. 1 to 5) and twocompounds (x4 and x22, N-benzyl-N-(cyclohexylcyclohexylcarbamoyl methyl)benzamide and N-(1-cyclohexylcarbamoyl pentyl) benzamide respectively)were characterised by Nuclear Magnetic Resonance Spectroscopy (NMR).

EXAMPLE 1B x4—Synthesis of x4

Using the general procedure of 1B above, to anhydrous methanol (0.5 ml)was added benzylamine (44 microlitre, 0.4 mmol) and cyclohexanecarboxaldehyde (48 microlitre, 0.4 mmol). The resulting solution wasagitated for 10 min at 28° C. To this solution was added ¹⁴C lightlyring labelled benzoic acid (from 1A) (48 mg, 0.4 mmol) in anhydrousmethanol (1 ml), followed by cyclohexylisocyanide (0.4 mmol). Thereaction was agitated for 60 h at 28° C., the resulting crudeprecipitate was filtered, washed with ice-cold methanol and dried undervacuum to yield the crude N-benzyl-N-(cyclohexylcyciohexyl carbamoylmethyl) benzamide (122 mg, 71%).

¹H NMR □ (400 MHz, CDCl₃) 7.55-6.85 (m, 10H), 4.67 (d, J 16.2 Hz, 1H),4.45 (d, J 16.2 Hz, 1H), 4.17 (d, J 9.5 Hz, 1H), 3.66 (m, 1H), 2.41 (m,1H), 1.95-1.45 (m, 10H), 1.40-0.85(m, 10H).

¹³C NMR □ (400 MHz, CDCl₃) 24.6, 25.5, 25.7, 25.7, 26.3, 29.7, 30.2,32.6, 32.9, 36.1, 47.7, 52.9, 66.7, 126.6, 127.2, 127.4, 128.2, 128.4,129,6, 136.7, 137.0, 169.2, 174.0

m/z (CI) 433 (M+H⁺); (found 433.2853, C₂₈H₃₇N₂O₂ requires for M+H⁺,433.2855)

1C—General Procedure for Solid-Phase Synthesis

Rink resin (1.1 mmol 0.055 g) was deprotected with 20% piperidine indichloromethane (DCM) (3×1 ml). The resin was swelled in 50% DCM:MeOH (1ml) for 30 min. Aldehyde (10 equiv. based on the initial resin loading)was added to the pre-swelled resin and the reaction mixture was agitatedfor 10 min at 28° C. ¹⁴C lightly ring labelled benzoic acid (from 1A)(10 equiv.) and isocyanide (10 equiv.) were added and resin was agitatedfor 36 h at 28° C. The resin was washed with DCM (10×5 ml) and driedunder vacuum. The resin was cleaved with 30% TFA:DCM (1 ml) for 3 h.Resin was removed by filtration and the filtrate was concentrated underreduced pressure to yield the crude condensation product.

EXAMPLE 1C x22—Synthesis of x22

Using the general procedure of 1C above, rink resin (1.1 mmol 0.055 g)was deprotected with 20% piperidine in dichloromethane (DCM) (3×1 ml).The resin was swelled in 50% DCM:MeOH (1 ml) for 30 min. Valeraldehyde(64 microlitre, 10 equiv. based on the initial resin loading) was addedto the pre-swelled resin and the reaction mixture was agitated for. 10min at 28° C. ¹⁴C lightly ring labelled benzoic acid (from 1A) (73 mg,10 equiv.) and cyclohexylisocyanide (76 microlitre, 10 equiv.) wereadded and resin was agitated for 36 h at 28° C. The resin was washedwith DCM (10×5 ml) and dried under vacuum. The resin was cleaved with30% TFA:DCM (1 ml) for 3 h. Resin was removed by filtration and thefiltrate was concentrated under reduced pressure to yield crudeN-(1-cyclohexylcarbamoyl pentyl) benzamide (17.9 mg, 94%).

¹H NMR □ (400 MHz, CDCl₃) 7.82 (d, J 7 Hz 2H), 7.54-7.42 (m, 3H),6.61(d, J 8 Hz, 1H), 4.63 (ddd, J 7,7,8 Hz), 3.77 (m, 1H), 2.0-1.5 (m,6H), 1.4-1.1 (m, 9H), 0.92-0.89(m, 3H)

¹³C NMR

□ (400 MHz, CDCl₃) 13.9, 22.4, 24.7, 25.4, 27.7, 32.3, 32.6, 32.8, 48.8,53.9, 127.2, 128.6, 132.0, 133.4, 167.6, 171.3. 117

m/z (CI) 317 (M+H⁺); (found 317.2227, C₁₉H₂₉N₂O₂ requires for M+H⁺,317.2229)

EXAMPLE 2 AMS Analysis of Library Samples

A sample of the ¹⁴C-benzoic acid precursor used in Example 1 and samplesof x4, x12, x19 and x22 obtained in Example 1 were graphitised using themethod of Vogel (Vogel J S (1992) Radiocarbon 34, 344-350) and analysedusing a NEC 15SDH-2 Pelletron AMS system. The terminal voltage was 4.5MV with a particle energy of approximately 22.5 MeV. At the centralterminal electrons were stripped from the carbon atom to yieldpositively. charged carbon ions (^(12, 13, 14)C^(+1 to +6)) C⁴⁺ ionswere selected for measurement as these are the most abundant at thisenergy.

The specific activity of the benzoic acid starting material was 1.56dpm/mg. The specific activities of the library compounds analysed wereas shown in Table 2. Compound Specific activity (dpm/mg) x4  0.58 x120.20 x19 0.25 x22 0.26

Thus all the library compound were all lightly labelled with ¹⁴C. Theoutput of an AMS is in units of “Percent Modern Carbon” (pMC). The meanbackground value was 2.37 pMC. Compounds X4 to X22 gave pMC values ofapproximately 2500, 3200, 2200 and 800 respectively. Thus although thespecific activities shown in Table 2 were low, they are well within thelimits of AMS measurement.

This indicates that the library compounds would be suitable formicrodosing to a subject in known manner to determine ADME and PK foreach compound.

EXAMPLE 2

A library of compounds having potential activity as antibacterials orbacteriophages are commercially available. 10 mg of each compound arelightly radio labelled by substitution with ¹⁴C to give a radioisotopelabelled library according to the invention. In this case the library issmall and the compound in each case is present in an independent viallabelled by library serial number and reference and the identity in eachcase is known by crossing the library serial number and reference with alibrary catalogue.

Compounds are screened to detect binding to a receptor moleculeassociated with the Salmonella microsome, using the Salmonella microsomeassay (Ames test). From the results a selection of candidate positivecompounds is made.

Candidate library compounds are already radioisotope labelled and maytherefore be forwarded directly for microdosing and AMS. The candidatecompounds are first made up in a form for microdosing each to adifferent human subject, in an amount of 5 microgram per subject. Afterseveral months samples of blood and urine are taken from each subjectand marked with the candidate library compound serial number. Samplesare prepared for AMS as known in the art. AMS is performed and resultsare analysed to indicate the metabolic characteristics of each candidatelibrary compound. From these a selection is made of candidates toforward for Stage I clinical trials, based on acceptable PKcharacteristics.

EXAMPLE 2

In this case a library of recombinant human antibodies is ¹⁴C labelledbiosynthetically using pooled essential ¹⁴C-amino acids. Sufficientradioactivity is incorporated to permit high limit of detection (severalthousand fold increase over ELISA 1.o.d) using AMS. The library isscreened for activity of individual radioisotope labelled libraryantibodies, by testing for receptor binding in a suitable receptorbinding assay and a selection is made for PK analysis. Microdosing iscarried out using prepared AMS samples of the selected candidate libraryantibodies using the method of Example 1, and studies are conducted inhuman serum spiked with the antibody and with rats administered theantibody. The method of the invention takes from 6 weeks to prepare theradioisotope labelled library (fairly independent on size of library inthis case as compounds may be radioisotope labelled in parallel by thebiosynthetic means described) and screen, identify candidate compoundsand conduct the AMS.

In a particular advantage, preclinical toxicology and clinical phasetrials may be performed on a candidate radioisotope labelled librarycompound of the invention, identified by the method of the invention.The entire process to completing clinical phase trials can be carriedout in 12 to 16 weeks.

The advantages of the radio labelled library of the invention and itsuse in the modified screening method of the invention are that thecandidate compound is synthesised only once in a microscale amount,there is no delay between screening and microdosing, shortening the timescale to identify an active drug candidate which offers the optimum PKcharacteristics for example, and therefore there is a greater certaintyfor start up and multinational drug discovery groups and investmentcompanies alike in basing a business plan around a candidate compound asa prospective pharmaceutical.

1-29. (canceled)
 30. Library of compounds or their pharmaceuticallyacceptable salts, provided as an array of compounds suitable for use inhigh throughput screening providing for in vitro activity, reactivity,inhibition or functionality screening and selection of compounds and inproviding binding data for the selected candidate compounds, by means ofAMS which can detect and quantify with relatively short analyticaltimes, levels of radioactivity that are so low that the dose needed tobe administered to a human subject falls below the stipulated levels ofradioactivity which require regulatory review, each compound beingassociated with information on its chemical identity and structure,wherein the library comprises a plurality of compounds or theirpharmaceutically acceptable salts of formula I:

wherein each

is different and is a compound which comprises an AMS activeradioisotope *; m is a value for the percent incorporation ofradioisotope which is a measure of maximum specific activity, wherein100% incorporation is defined as the incorporation of one radioisotopeper molecule, taking a given amount of substance, in which everymolecule has one specified atom replaced with its radioactive equivalent; and t is a tag associated with information on the compounds chemicalidentity and structure wherein n is 0, or a whole number integer whereinm is in the range from in excess of zero to 0.1% whereby each compoundof formula I is lightly labelled, a proportion thereof having noradioisotope, characterised in that the library is a chemical library.31. Library as claimed in claim 30 for use in candidate compoundselection, detecting the amount of compound and optionally the locationin a sample from screening, using AMS radiodetection techniques. 32.Library as claimed in claim 30 wherein an array is a screening plate,microtiter plate, cell array, vial or bottle array, support matrix orplate or fibre optic array.
 33. Library as claimed in claim 30 whereinan array is a spatial array, wherein compounds are distributed over ahoneycomb plate, with each well in the honeycomb having 0 or 1 compound.34. Library of compounds as claimed in claim 30 wherein percentincorporation (m) is in the range 1×10⁻¹² to 0.1%.
 35. Library asclaimed in claim 30 wherein an AMS active radioisotope is selected fromany one or more of ²H, ³H, the isotopes of Ba, ¹⁰Be, ¹⁴C, ¹⁷O, ¹⁸O,²⁶Mg, ²⁶Al, ³²Si, ³⁶Cl, ⁴¹Ca, ⁵⁵Fe, ⁵⁷Fe, ⁶⁰Fe, ⁵³Mn, ⁵⁵Mn, ⁷⁹Se and¹²⁹I, ²³⁶U and ²³⁹Pu.
 36. Library as claimed in claim 35 wherein an AMSactive radioisotope is a ¹⁴C radioisotope, optionally additionally a³⁶Cl or ³H radioisotope.
 37. Library as claimed in claim 30 comprisingcompounds in quantities of nanomoles or millimoles, typicallymilligrams, by virtue of the sensitivity of AMS detection techniques.38. Process for the preparation of a library of compounds as claimed inclaim 30 comprising AMS active radioisotope labelling a plurality ofcompounds, each compound being associated with information on itschemical identity and structure wherein labelling is conducted as partof any single or multistep synthetic route which comprises labelling asynthetic precursor or intermediate, determining the specific activitythereof, determining the desired specific activity to give a desiredpercent incorporation, and combining with a sufficient amount ofcorresponding unlabeled synthetic precursor or intermediate andisolating as a homogeneous product having desired percent incorporation.39. Method for selecting one or more candidate compounds comprising highthroughput screening a library of the invention comprising AMS activeradioisotope labelled compounds as hereinbefore defined in claim 30 andobtaining samples from the screen, and performing AMS detection of thesamples and thereby detecting and quantifying the amount and optionallythe location of candidate compound in each sample.
 40. Method as claimedin claim 39 comprising providing in vitro activity, reactivity,inhibition or functionality screening and selection of compounds and inproviding binding data for the selected candidate compounds.
 41. Methodas claimed in claim 39 wherein a sample for AMS detection is derivedfrom a screen, such as a cell or cell membrane sample.
 42. Method asclaimed in claim 39 wherein screening is a human or animal biomedicalassay or the like, for example a protein binding assay, such as areceptor binding assay.
 43. Method for AMS detection and quantificationof AMS active radioisotope obtained from one or more radioisotopelabelled compounds present in screening samples using the method asclaimed in claim
 39. 44. Use of a library or a method as hereinbeforedefined in claim 30 in (bio)medical, agrochemical, environmental andlike screening for further study and quantification by AMS detection.45. Use as claimed in claim 44 wherein (bio)medical screening is forcompound activity, reactivity such as binding, inhibitory effect;agrochemical screening is for compound activity, reactivity such asbinding, inhibitory effect or assessing for plant, insect or likemetabolism; environmental screening is for compound activity, reactivitysuch as binding, inhibitory effect or other functionality, and assessingfor soil, aqueous or sediment absorption or adsorption, diffusion,leaching, metabolism, degradation, dissipation or photolysis study. 46.Use as claimed in claim 44 for detecting the amount of a compound in anylocation or sample, using AMS radiodetection techniques.